Construction of Genetically Encoded Biosensors to Monitor Subcellular Compartment-Specific Glutathione Response to Chemotherapeutic Drugs in Acute Myeloid Leukemia Cells

Glutathione (GSH), the constituent of the redox buffer system, is a scavenger of reactive oxygen species (ROS), and its ratio to oxidized glutathione (GSSG) is a key indicator of oxidative stress in the cell. Acute myeloid leukemia (AML) is a highly aggressive hematopoietic malignancy characterized by aberrant levels of reduced and oxidized GSH due to oxidative stress. Therefore, the real-time, dynamic, and highly sensitive detection of GSH/GSSG in AML cells is of great interest for the clinical diagnosis and treatment of leukemia. The application of genetically encoded sensors to monitor GSH/GSSG levels in AML cells is not explored, and the underlying mechanism of how the drugs affect GSH/GSSG dynamics remains unclear. In this study, we developed subcellular compartment-specific sensors to monitor GSH/GSSG combined with high-resolution fluorescence microscopy that provides insights into basal GSH/GSSG levels in the cytosol, mitochondria, nucleus, and endoplasmic reticulum of AML cells, in a decreasing order, revealing substantial heterogeneity of GSH/GSSG level dynamics in different subcellular compartments. Further, we investigated the response of GSH/GSSG ratio in AML cells caused by Prussian blue and Fe3O4 nanoparticles, separately and in combination with cytarabine, pointing to steep gradients. Moreover, cytarabine and doxorubicin downregulated the GSH/GSSG levels in different subcellular compartments. Similarly, live-cell imaging showed a compartment-specific decrease in response to various drugs, such as CB-839, parthenolide (PTL), and piperlongumine (PLM). The enzymatic activity assay revealed the mechanism underlying fluctuations in GSH/GSSG levels in different subcellular compartments mediated by these drugs in the GSH metabolic pathway, suggesting some potential therapeutic targets in AML cells.


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Cell culture
The human leukemia cell line HL60 and human embryonic kidney (HEK) 293T cells were cultured and maintained in RPMI 1640 medium (Gibco, USA) and DMEM (Gibco, USA), respectively, supplemented with 10% fetal bovine serum (FBS, Gibco, USA)) and 1% penicillinstreptomycin (PS, Life Technologies). All the cells were cultured in humidified air containing 5% CO 2 at 37 °C. 293T cells were kindly gifted by Dr. Guang-Xia Gao (Institute of Biophysics, Chinese Academy of Sciences), while the HL60 cell line was obtained from Huiying-Bio (Shanghai, China).

Sensor plasmids cloning
The primers for PCR amplification, subcellular-targeting sequences, and sensor encoding sequences of Grx1-roGFP2 and Grx1-roGFP2.iL were synthesized by Sangon Biotechnology mitochondrial (MLS-Grx1-roGFP2) expression, respectively. The N-terminus of the Grx1-roGFP2.iL encoding sequence was fused with the ER localization sequence similar to that of the pCMV/myc/ER plasmid and at the C-terminal ER retention signal (KDEL) for expression in ER.
The designed plasmids were subcloned into the pLVX lentiviral vector backbone for expression in mammalian cells. All the constructs were confirmed via DNA sequencing.

Live-cell imaging of subcellular compartments
The AML stable cell lines were stained with Hoechst 33342 (

Characterization of the GSH/GSSG sensor in AML stable cell lines
The

Cell viability and proliferation assay
The CCK-8 assay (Beyotime, Shanghai, China) was used to determine cell viability according to the manufacturer's instructions. Cells were seeded in a 96-well microplate (10 4 cells/well) along with the aforementioned chemical treatments. Subsequently, 10 μL of CCK-8 solution was added to each well and incubated for 2 h at 37 °C in humidified air containing 5% CO 2 . The absorbance was measured at 450 nm using a microplate reader (BioTek, USA).

Enzymatic assays and molecular docking
The

Statistical analysis
Data are presented as the mean ± standard deviation (SD) unless otherwise stated, and all the experiments were independently repeated at least three times. Statistical analyses were S-6 performed using OriginPro and GraphPad Software. One-way analysis of variance (ANOVA) was used to compare differences between different groups (****P<0.0001, ***P<0.001,**P<0.01,*P<0.05).

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Additional Results Figure S1. Panels (A, B, and C) represent the Pearson's correlation coefficient (PCC) between the glutathione probe signal and the tracking dye.

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Scale bar: 20 μm. (G) Cell viability of NMM treated HL60 cells after 24 h, whereas the treatment group is significantly different from the control group. One-way ANOVA was used to compare the differences between different groups. Data are presented as the mean ± SD, n≥3.

Effect of NPs alone or in combination with Ara-C on GSH/GSSG levels in AML cells.
To study the effect of NPs on AML cells, the Cyto-Grx1-roGFP2-expressing stable cell line was respectively treated with PBNPs and Fe 3 O 4 NPs at different concentrations for 24 h.
Moreover, these two NPs were added to the Cyto-Grx1-roGFP2-expressing stable cell line combined with 1 μM Ara-C to investigate the collective effect of NPs and chemotherapeutic drugs.
This demonstrated that PBNPs caused an increase in the GSH/GSSG levels in AML cells in a concentration-dependent manner ( Figure S3A). While Ara-C impaired the GSH/GSSG levels induced by PBNPs ( Figure S3B). The response ratio showed that Fe 3 O 4 NPs downregulated the GSH/GSSG levels in AML cells in a concentration-dependent manner ranging from 0 to 100 μg/mL ( Figure S3C), which displayed a synergistic effect with Ara-C and further decreased the GSH/GSSG levels ( Figure S3D). with Ara-C after 24 h determined using flow cytometry (≥ 10, 000 cells). Bandpass 530/30 nm and 525/50 nm emission filters were used for 488 nm and 405 nm excitation wavelengths, respectively. One-way ANOVA was used to compare the differences between different groups. Data are presented as the mean ± SD, n≥3.
S-10 GSH metabolic pathway Figure S4. Glutathione metabolic pathway and varied distribution of various enzymes in distinct subcellular compartments.